High-throughput evaluation of the critical micelle concentration of detergents

Determination of the critical micelle concentration (CMC) value of detergents routinely used in biological applications is necessary to follow possible changes due to different buffer compositions (e.g., temperature, pH) such as those in solutions that are used for protein activity assays or crystallization. Here we report a method to determine the CMC values of detergents through a fast and robust assay that relies on the fluorescence of Hoechst 33342 using a 96-well plate reader. Furthermore, this assay provides the possibility and sensitivity to measure the CMC of detergent mixtures. The examples described here emphasize the potential and applicability of this assay and demonstrate that analysis of the physicochemical parameters of detergents can now be investigated in virtually every laboratory.     

Effect of anionic micelles on speciation of L-glutamine in presence/absence of metals

Abstract

Protonation equilibria of L-glutamine and speciation of its complexes with Co(II), Ni(II) and Cu(II) have been studied in 0–2.5% w/v SLS–water media using pH-metric method. The protonation constants and binary stability constants have been calculated with the computer program MINIQUAD75. Selection of the best fit chemical models is based on standard deviation in stability constants and residual analysis using crystallographic R-factor and sum of squares of residuals in all mass-balance equations. The trend in the variation of stability constants of the complexes with mole fraction of the medium is attributed to the compartmentalisation of complexation equilibria. Distribution of species and effect of influential parameters on chemical speciation have also been presented.     

Phytosterol ester processing in the small intestine: impact on cholesterol availability for absorption and chylomicron cholesterol incorporation in healthy humans

Phytosterols (plant sterols and stanols) can lower intestinal cholesterol absorption, but the complex dynamics of the lipid digestion process in the presence of phytosterol esters (PEs) are not fully understood. We performed a clinical experiment in intubated healthy subjects to study the time course of changes in the distribution of all lipid moieties present in duodenal phases during 4 h of digestion of meals with 3.2 g PE (PE meal) or without (control meal) PE. In vitro experiments under simulated gastrointestinal conditions were also performed. The addition of PE did not alter triglyceride (TG) hydrolysis in the duodenum or subsequent chylomicron TG occurrence in the circulation. In contrast, cholesterol accumulation in the duodenum aqueous phase was markedly reduced in the presence of PE (-32%, P < 0.10). In vitro experiments confirmed that PE reduces cholesterol transfer into the aqueous phase. The addition of PE resulted in a markedly reduced presence of meal-derived hepta-deuterated cholesterol in the circulation, i.e., in chylomicrons (-43%, PE meal vs. control; P < 0.0001) and plasma (-54%, PE meal vs. control; P < 0.0001). The present data show that addition of PE to a meal does not alter TG hydrolysis but displaces cholesterol from the intestinal aqueous phase and lowers chylomicron cholesterol occurrence in humans.     

Cutinase-AOT interactions in reverse micelles: the effect of 1-hexanol

Cutinase encapsulated in dioctyl sulfosuccinate reverse micelles displays very low stability, undergoing fast denaturation due to an anchoring at the micellar interface. The denaturation process and the structure of the reverse micelle were characterized using biophysical techniques. The kinetics of denaturation observed from fluorescence match the increase of the hydrodynamic radius of reverse micelles. Denaturation in reverse micelles is mainly the unfolding of the three-dimensional structure since the decrease in the circular dichroism ellipticity in the far-UV range is very small. The process is accompanied by an increase in the steady-state anisotropy, as opposed to what happens for denaturation in aqueous solution.Since 1-hexanol used as co-surfactant in dioctyl sulfosuccinate reverse micelles slows or even prevents cutinase denaturation, its effect on cutinase conformation and on the size of reverse micelles was analyzed. When 1-hexanol is present, cutinase is encapsulated in a large reverse micelle, as deduced from dynamic light scattering. The large reverse micelle filled with cutinase was built from the fusion of reverse micelles according to a pseudo-unimolecular process ranging in time from a few minutes to 2h depending on the reverse micellar concentration. This slow equilibrium driven by the encapsulated cutinase has not been reported previously. The encapsulation of cutinase in dioctyl sulfosuccinate reverse micelles establishes a completely new equilibrium characterized by a bimodal population of empty and filled reverse micelles, whose characteristics depend greatly on the interfacial characteristics, that is, on the absence or presence of 1-hexanol.     

Micellar oleic and eicosapentaenoic acid but not linoleic acid influences the β-carotene uptake and its cleavage into retinol in rats

Improving the bioavailability of β-carotene is vital to manage vitamin A deficiency. The influence of micellar oleic (OA), linoleic (LA) and eicosapentaenoic (EPA) acids on plasma β-carotene response and its conversion to retinol has been studied in rats employing single (9 h time course) and repeated (10 days) dose administrations. After a single dose, the levels (area under the curve) of plasma β-carotene and retinyl palmitate in OA and EPA groups were higher (p < 0.05) by 13, 7 and 11, 6 folds than LA group. The liver β-carotene level in OA and EPA groups were higher (p < 0.05) by 3 and 1.2 folds than LA group. After repeated dose, the plasma β-carotene and retinyl palmitate levels in OA (6.2%, 51.7%) and EPA (25.4%, 17.23%) groups were higher (p < 0.05) than LA group. The liver β-carotene level in OA (21.2%) and EPA (17.6%) groups were higher (p < 0.05) than LA group. In both the experiments, the activity of β-carotene 15,15′-dioxygenase in the intestinal mucosa and plasma triglyceride levels were also higher in OA and EPA groups than LA group. β-Carotene excreted through urine and feces of OA and EPA groups was lower than the LA group. These results demonstrate an improved absorption and metabolism of β-carotene when fed mixed micelles with OA or EPA compared with LA. Although the mechanism involved in selective absorption of fatty acids needs further studies, intestinal β-carotene uptake and its conversion to vitamin A can be modulated using specific fatty acids.     

D-甘露糖修饰聚合物胶束的制备及其在靶向药物输送中的应用

聚合物胶束作为药物载体具有良好的稳定性和生物相容性,提高疏水性药物溶解性等优势,是一类很有应用潜力的药物传输系统。本研究以合成的共价键连D-甘露糖的双亲性聚合物分子(PGMA-Mannose)为药物载体,包载抗癌药物阿霉素(DOX)制备具有甘露糖受体靶向性和pH敏感药物释放特性的新型载药聚合物胶束。利用激光共聚焦显微镜和MTT细胞毒性评价方法对载药胶束的细胞内吞摄取和毒性进行评价。实验结果表明,载药胶束能特异性识别人乳腺癌细胞MDA-MB-231表面过度表达的甘露糖受体,被癌细胞大量摄取并在细胞溶酶体酸性环境内释放药物,而载药胶束在表面甘露糖受体低表达的HEK293细胞中只有少量摄取。与原药DOX相比,该载药胶束对癌细胞的毒性显著提高,而对正常细胞的毒性较低。因此,该PGMA-Mannose聚合物胶束有望成为一种新型的靶向药物输送系统应用于癌症的治疗。     

The effect of demulsifiers on lysozyme extraction from hen egg white using reverse micelles

The liquid–liquid extraction of protein from buffered aqueous phases using reverse micelles (RM) has been extensively researched from a fundamental point of view. However, very little effort has been expended at scaling up this process for the extraction of real fermentation broth. When real broths are used with reverse micellar phases there are major problems with emulsion formation. In this study the effect of a variety of demulsifiers on lysozyme extraction was evaluated in terms of their influence on the separating properties of the emulsion, water content (W _o ), and, extraction yield and kinetics from both buffer and hen egg white. In addition, the use of a low shear contactor (a Graesser or `raining bucket') was assessed in terms of its suitability as a RM contactor. It was found that most of the demulsifiers reduced the settling time of the emulsion, and enhanced the yield and kinetics of lysozyme extraction from hen egg white. It was hypothesised that this was due to the demulsifier displacing the lysozyme from the interface and preventing the protein unfolding and precipitating. This effect was found to depend on both the generic type of demulsifier, and its concentration.     

Kinetics of the photosensitized oxidation of chymotrypsin in different media

Summary. The kinetic aspects of the Perinaphthenone-sensitized photooxidation (singlet molecular oxygen [O₂ (¹Δ_g)]-mediated) of α-chymotrypsin (α-Chymo) have been studied at pH 8 and pH 11 as well in reverse micelles (RMs) of sodium 1, 4 bis (2-ethylhexyl) sulfosuccinate (AOT) in n-heptane. The rate constant values for both overall (k_t) and chemical (k_r) quenching of O₂ (¹Δ_g) by α-Chymo in homogeneous media were higher at pH = 11 than at pH = 8, indicating that the OH-ionized tyrosine (Tyr) residues, clearly dominate the quenching process. Besides, the rate constants in water were higher than those determined in RMs, demonstrating that the organized medium protects the protein against photooxidation, probably due to a diminution in both, the accessibility towards oxidizable amino acid residues and the polarity inside the aggregate, as compared to water. The protection effect of α-Chymo against the attack by the oxidative species O₂ (¹Δ_g) in RMs of AOT seems to be due to the increase of protein stability by the encapsulation within the micellar structure. The effect of both, surfactant concentration and variation of the ratio ([H₂O]/[AOT]) = W on the reactive rate constant was also investigated. The process does not depend significantly on micelles concentration while the k_r values increase as W increases. Furthermore, at W = 30, the highest W studied, k_r tends to the value obtained in aqueous medium. Authors’ address: M. A. Biasutti, Departamento de Química, Campus Universitario, Universidad Nacional de Río Cuarto, (X5804ALH) Río Cuarto, Argentina     

A coarse-grained model for PCL: conformation,self-assembly of MePEG-b-PCL amphiphilic diblock copolymers

Poly-ε-caprolactone (PCL) is a biodegradable hydrophobic polyester that has been widely used in medical devices, tissue engineering and nanoparticle-based drug delivery. Coarse-grained molecular dynamics (CGMD) has been employed to study and gain insights into the conformational, structural and self-assembly behaviour of polymers, lipids and amphiphilic macromolecules. In this work, we developed a model for PCL within the framework of the MARTINI coarse-grained force field. The non-bonded interactions were based on the existing MARTINI bead types, while the bonded interactions were mapped onto a PCL rendition obtained from atomistic simulations. The model accurately reproduces the structural and dynamic properties of the PCL homopolymer and shows very reasonable temperature and solvent transferability. We also studied self-assembly of MePEG-b-PCL linear diblock copolymers using an existing MARTINI model for MePEG (Methoxy Polyethylene glycol), by analysing the critical micelle concentration (CMC), as well as the shape, size and morphology of the nano-polymeric micelles. We obtained excellent agreement of the CMC, while the size was under-predicted compared to experimental data. This robust model paves the way for CGMD modelling of PCL and serves as a starting point for future designs of PCL-related polymeric systems .     

Dipeptide synthesis and separation in a reversed micellar membrane reactor

The synthesis of dipeptide AcPheLeuNH₂ catalyzed by α-chymotrypsin encapsulated in TTAB/octanol/heptane reversed micelles was investigated in a tubular ceramic membrane reactor, operated in a batch mode. The reaction medium conditions (TTAB concentration, buffer molarity, and pH) were optimized using a factorial design in order to achieve maximum synthesis rates. Hydrated reversed micelles permeated through the membrane together with the substrate ester, dipeptide, and by-products. However, as a result of the low solubility of the peptide in the reaction medium, selective precipitation occurred, thus enabling the complete retention of the solid product by the ultrafiltration membrane and therefore an integration of a separation step in the biotransformation process. In spite of the continuous accumulation of solids inside the reactor, constant permeation flow rates could be maintained throughout the operation. The influence of α-chymotrypsin, TTAB, and water concentration on the kinetics and mass transfer of the system was also investigated. The behavior of the system during a continuous experiment was also evaluated.